Stefan Ehlers

Tumor necrosis factor and its blockade in granulomatous infections : Differential modes of action of infliximab and etanercept?

Tumor necrosis factor (TNF) is a critical component of both the antibacterially protective and the inflammatory responses against infections, particularly infections with intracellularly viable microorganisms. It is, therefore, not surprising that some treatment regimens that target TNF function have resulted in an increase in complications associated with infections due to such pathogens as Mycobacterium tuberculosis, Listeria monocytogenes, and Histoplasma capsulatum; organized granuloma formation is required to keep such infections under control. However, treatment with anti-TNF monoclonal antibodies (i.e., infliximab) has been associated with a higher incidence of granulomatous infections than has treatment with a TNF receptor (TNFR) p75 immunoglobulin G-fusion construct (i.e., etanercept). Three hypotheses concerning the mode of action of these 2 agents that might explain this difference are discussed here: differential induction of apoptosis or lysis in membrane TNF-expressing macrophages and T cells, differential inhibition of signaling via TNFRp55 and TNFRp75, and different net neutralizing capacities resulting from different pharmacologic properties.

The Role of Cytokines in Experimental Listeriosis

Listeria monocytogenes is a Gram-positive, intracytoplasmatically replicating pathogen that elicits host reactions which are very similar in man and rodents. Using murine listeriosis as a highly reproducible and convenient experimental model for studying the immune response to infections with facultative intracellular bacteria, Mackaness developed the concept of T cell-mediated macrophage activation as the pivotal mechanism in host defense against this type of infectious agents. Continued research in listeriosis itself, however, provided paradoxical findings that challenged the original dogma. In particular, the finding that T cell-mediated inflammatory events, like DTH and granuloma formation, can be dissociated from protective effector mechanisms has provided a new impetus and experimental access to characterizing the molecular mediators responsible for these diverging phenomena. This review first summarizes the cellular basis for the dichotomy of immunological phenomena outlined above and will then relate recent findings on cytokine expression in infected tissues to these dual categories of the host response to infection. The authors will focus on data obtained from in vivo experiments and draw on evidence from in vitro systems only when appropriate in vivo verification is still lacking. The data presented will cover the developments made in the field of cytokine research since our previous review in 1981 (Rev. Infect. Dis. 3: 1221-1250). Detectable numbers of listeria-specific T cells become apparent on day 4 to 5 of a primary infection. Whereas the localized and sustained release of TNF and IFN-gamma mediated by CD4+ cells seems to be the focusing event triggering mononuclear cell accumulation, the coincidental eradication of bacteria critically depends on CD8+ and/or CD4-CD8-Thy1+ cells. Their effector functions, however, remain obscure, since cytokines cannot be identified that will substitute for their presence. None of the cytokines studied thus far has been demonstrated to effectively cure an established infection. In addition, the increased production of cytokines characteristic of an anamnestic response (IL-2, IL-3, IL-4, IFN-gamma and TNF) can be dramatically reduced by depleting CD4+ T cells without any effect on the animals ability to eradicate high lethal doses of bacteria and Listeria-specific CD8+ T cells can mediate protection even in the presence of neutralizing antibodies to IFN-gamma. In conclusion, the murine model of Listeria infection provides an interesting experimental approach for the development of immunotherapeutic strategies aimed at reducing T cell-mediated immunopathology without interfering with innate resistance and T cell-mediated cure and prevention of disease.

Infection of the Skin Caused by Corynebacterium ulcerans and Mimicking Classical Cutaneous Diphtheria

Extrapharyngeal infections caused by Corynebacterium ulcerans have rarely been reported previously, and diphtheria toxin production has usually not been addressed. This case demonstrates that strains of C. ulcerans that produce diphtheria toxin can cause infections of the skin that completely mimic typical cutaneous diphtheria, thereby potentially providing a source of bacteria capable of causing life-threatening diseases in the patients environment. Therefore, it is recommended to screen wound swabs for coryneform bacteria, identify all isolates, carefully assess possible toxin production, and send questionable strains to a specialist or a reference laboratory.

Surface-modified amikacin-liposomes : Organ distribution and interaction with plasma proteins

Amikacin-loaded liposomes were produced and surface-modified by adsorption of PEG 4000, Tween 80, poloxamer 407 and gelatin. The organ distribution was studied in mice by analysing the amikacin content in liver, spleen, lung, kidneys and serum. Highest serum levels were obtained with the PEG- and Tween 80 modified liposomes (at 2 hours p.inj.). Modification of the liposomes with gelatin as opsonization promoting agent distinctly increased the amikacin concentration in the liver from 36 to 66 mg/kg. Highest spleen concentrations were observed with non-modified and poloxamer 407 liposomes (242 mg/kg and 248 mg/kg, respectively). The data suggest that modification by a simple adsorption process is sufficient to effectively alter the organ distribution. The liposomes differing in organ distribution exhibited also different plasma protein adsorption patterns, up to 115 spots were detected by 2-D PAGE. Hydrophilic albumin was present in a conc. of appr. 80% on liposomes modified with ethoxylated compounds. On the gelatin liposomes, 14% of alpha-2-Macroglobulin were adsorbed which is a protein typically found on particles rapidly cleared by the RES. IgM, Apo A-I, Apo C-II and alpha-1-Antitrypsin were other detected proteins.

Liposomal Amikacin for Treatment of M. avium Infections in Clinically Relevant Experimental Settings

In an effort to optimize rational chemotherapy against M. avium infections in a clinically meaningful context, we tested whether liposome-encapsulated amikacin would effectively reduce the bacterial load in (i) intravenously infected immunodeficient SCID mice, (ii) immunocompetent mice in both early and late stages of intravenous infection, and (iii) immunocompetent mice with pulmonary M. avium infection. Although complete eradication of M. avium was never achieved following intravenous infection, mycobacterial CFUs decreased by 3 to 4 logs in the spleens and livers of mice treated for three weeks with twice-weekly intravenous injections of liposomal amikacin and continued to stay low in the liver, even in the absence of specific immunity. Mice treated in the chronic stage of infection equally benefited from therapy and showed signs of attenuated granulomatous inflammation in the liver. Even moribund mice responded to liposomal amikacin by significantly gaining weight and survived their infected untreated littermates by at least 4 months. In contrast, during pulmonary M. avium infection, treatment with liposome-encapsulated amikacin only resulted in a transient plateau of bacterial proliferation in the lungs, and the infection exacerbated immediately after cessation of therapy.

The role of T cell subpopulations in cell mediated immunity to facultative intracellular bacteria

SummaryThis brief review summarizes the experimental data which underly the classic concept of anti-bacterial cell mediated immunity and will integrate recent developments focusing on results obtained byin vivo studies in the model of rodent listeriosis.ZusammenfassungDiese kurze Übersicht faßt die experimentellen Daten zusammen, die dem klassischen Konzept antibakterieller zellvermittelter Immunität zugrunde liegen, und schließt die neuesten Erkenntnisse unter besonderer Berücksichtigung vonIn-vivo-Untersuchungen am Modell der Listeriose von Maus und Ratte ein.

HPLC determination of clofazimine in tissues and serum of mice after intravenous administration of nanocrystalline or liposomal formulations

A simple HPLC method is described for the determination of clofazimine in mouse tissues and in serum. The main application of the method was the determination of the drug in mouse tissues after i.v. administration of nanocrystalline suspensions or liposomal encapsulated clofazimine. Tissues were extracted with a 10-fold (w/v) volume of an extraction solution consisting of methanol/glacial acetic acid 9:1 (v/v). Serum proteins were precipitated with a 2-fold volume of acetonitrile. Isocratic chromatography was performed using an anion exchange column (Nucleosil 100-5 SA, Macherey & Nagel) for separation. The mobile phase was a mixture of acetonitrile and 0.1 mol/l aqueous phosphoric acid (75:25, v/v), adjusted to pH 2.9 with sodium hydroxide solution. Absorption of the eluate was monitored at 495 nm. The assay was precise, simple to perform and fast. Recovery from tissues was > or = 98%, from nanoparticles > or = 98%, and from liposomes > or = 96%. No interference was observed in extracts from mouse liver, spleen, lungs and human serum.

The mRNA-Phenotype of Granuloma Formation: CD4+ T Cell-Associated Cytokine Gene Expression during Primary Murine Listeriosis

In murine listeriosis, elimination of bacteria and immunity to reinfection critically depend on Thy1+ CD4- cells, while cell-mediated inflammatory phenomena such as DTH and granuloma formation are mostly mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced, we examined the cytokine gene expression profile associated with the presence or absence of Thy1+, CD4+ and/or CD8+ cells in the livers of mice during a primary infection with L. monocytogenes. The presence of CD4+ cells was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of TNF-alpha and GM-CSF and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, and temporally coincided with the development of granulomatous lesions. In vivo neutralization of TNF-alpha and, to a lesser extent, IFN-gamma resulted in abrogation of granuloma formation. A similar correlation between the presence of CD8+ cells and mRNA expression for any one of the cytokines studied did not exist, pointing to a qualitatively different mechanism of CD8+ T cell mediated cure of listeriosis.

The influence of ciprofloxacin treatment in vivo on cell-mediated immunity to Listeria monocytogenes

The intravenous and intraperitoneal administration of ciprofloxacin in very high doses (2 x 1 mg/d up to 2 x 2 mg/d) can reduce the bacterial load of mice experimentally infected with the intracellular bacterium, Listeria monocytogenes. The T-cell response generated during the treated infection is affected in much the same way as it is during antibiotic treatment with ampicillin, i.e. the protective immunity established directly correlates with the number of bacteria present during an extended period of time during the primary infection. Although an additional anti-proliferative effect of ciprofloxacin on expanding T-cells as evidenced in in vitro experiments cannot be excluded, our data in summary favour the view that in vivo this effect is at most of minor importance.

Effects of Staphylococcus epidermidis on Cellular Immunity to Infection with Listeria monocytogenes

With the present study, the effects of intravenous applications of Staphylococcus epidermidis (SE) on the course of experimental infections of mice with Listeria monocytogenes were evaluated. SE treatment 24 h prior to Listeria infection led to a reduced growth of Listeria organisms in both livers and spleens and to an increased resistance of infected animals against a lethal Listeria challenge. SE treatment 24 h after Listeria infection resulted in an enhanced growth of and retarded elimination of Listeria organisms from animal organs as well as in a reduction of delayed-type hypersensitivity to soluble Listeria antigen. Adoptive immunotherapy accomplished by transferring immune peritoneal exudate T-lymphocyte-enriched cells (PETLEs) to Listeria-infected recipients 24 h before SE treatment did not prevent the delay in clearance of Listeria organisms. When Listeria-infected recipients compromised in their immune response by SE treatment were infused with immune PETLEs either immediately or 24 h after the application of SE, the immunosuppression induced by SE proved to be reversible. It is concluded that, in analogy to other bacterial immunomodulators, Staphylococcus epidermidis is able to either nonspecifically activate macrophages or interfere with T-lymphocyte functions.